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The cytoskeleton: from regulation to function

2000; Springer Nature; Volume: 1; Issue: 6 Linguagem: Inglês

10.1093/embo-reports/kvd123

1469-3178

Anthony Bretscher,

3D Printing in Biomedical Research

Meeting Report1 December 2000Open Access The cytoskeleton: from regulation to function Conference: The 15th Meeting of the European Cytoskeleton Forum Anthony Bretscher Corresponding Author Anthony Bretscher Search for more papers by this author Anthony Bretscher Corresponding Author Anthony Bretscher Search for more papers by this author Author Information Anthony Bretscher *Corresponding author. Tel: +1 607 255 5713; Fax: +1 607 255 6249; E-mail: [email protected] EMBO Reports (2000)1:473-476https://doi.org/10.1093/embo-reports/kvd123 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The European Cytoskeleton Forum (formerly the European Cytoskeleton Club) was established 20 years ago by G. Gabbiani (chair), W.W. Franke, B. Geiger, B.M. Jockush, U. Lindberg, M. Osborn, F. Ramaekers, J.V. Small, J. Vandekerckhove and K. Weber. The Forum now has an annual meeting in an attractive European location to highlight the latest progress in cytoskeleton research. The 15th Meeting of the European Cytoskeleton Forum, held in Blankenberge on the Belgian coast at the end of August, was attended by about 160 participants from Europe, with a sprinkling of speakers form the USA and Japan. The outstanding program was superbly organized by joël Vandekerckhove, Christophe Ampe and Jan Gettemans (Ghent, Belgium). The meeting was sponsored by Euroconference grant No. ERBFMMACT980455, and by grant WO.028.00N from the Fund for Scientific Research-Flanders (Belgium). Introduction How the field has changed! In the early days of cytoskeletal research, intense interest was generated by the identification and cataloging of new cytoskeletal proteins, and their grouping into functional classes, which now number >60. While this important first step continues, and will surely be expanded as mammalian genomes are completed, the field has also advanced to other levels. This has resulted in the emergence of an integrated view of how signal transduction pathways regulate cytoskeletal elements that participate in cell structure, motility and membrane traffic. Since no meeting can cover such a large area in great depth, it is necessary for the organizers to select an area for emphasis, preferably spiced with major advances in related topics. The 15th Meeting of the Cytoskeletal Forum did this admirably, with an emphasis on microfilament regulation and function. Here I attempt to provide an overview of the central focus of the meeting, rather than provide a comprehensive account, so many interesting contributions are not mentioned. Regulation by Rho GTPases The central theme of the meeting was ably articulated by keynote speaker A. Hall (London, UK) who summarized his pioneering work on microfilament regulation by the Rho family of small G proteins. The field was founded some years ago when it was demonstrated that, in quiescent cells, activation of Cdc42 induces filopodia, activation of Rac induces lamellipodia, and activation of Rho induces stress fiber formation. This background set the stage for analyzing the specific roles of, and cooperation between, these regulatory proteins as well as components of downstream pathways, in a wide spectrum of microfilament-based processes. For example, the distinct roles played by Rho GTPases can be determined by analyzing effects on migration after wounding a cell monolayer. Thus, Hall described studies showing that Cdc42 is necessary for polarity determination, that Rac regulates lamellipodial protrusion, and that Ras, acting through Rho, regulates focal adhesion turnover. The roles played by Rho GTPases within a particular cell can also depend on specific ligands capable of inducing phenotypically similar responses. For example, Hall described how CD3-mediated induction of phagocytosis in macrophages requires activation through Rho but not Rac or Cdc42, whereas induction through the Fcγ receptor requires activation through Rac and Cdc42 but not Rho. This theme was extended by J. Collard (Amsterdam, The Netherlands) who discussed the effects of manipulating the ratio of active Rac/Rho on integrin-dependent cell morphology and cell migration. Fibroblasts transfected with the Rac activator Tiam1 exhibit both enhanced spreading and E-cadherin-based cell–cell adhesion, as well as a reduced level of active Rho, to yield an epithelial-like phenotype. In contrast, activation of Rho enhances contractility and cell-substrate interactions to give a mesenchymal migratory phenotype. The universal importance of Rho GTPases was further illuminated by E. Lemichez (New York, NY) who described the role that abscisic acid plays in the plant Arabidopsis, inactivating Rac and disassembling the actin cytoskeleton to induce guard cell relaxation for stomatal closure. Additional regulatory pathways Class IA phosphoinositide 3-kinases (PI3Ks) are also important regulators. These lipid kinases are activated by receptor tyrosine kinases, which have specific phospho-tyrosine residues that bind to the SH2 domains of the p85 regulatory subunits of PI3Ks. This causes the activation of the 110 kDa catalytic subunits, which can, in some cases, activate the exchange factors of the Rho GTPases. B. Vanhaesebroeck (London, UK) discussed the distinct contributions of different PI3K 110 kDa isoforms to cellular processes. Catalytically similar enzymes are encoded by three different genes. The p110α and β forms are ubiquitously expressed, and the p110δ form is found mainly in leukocytes. Interestingly, in cytokine-stimulated macrophages, p110α controls cell proliferation whereas the p110β and p110δ forms regulate the actin cytoskeleton and cell migration. A clear example of parallel signaling was provided by J. Sturge (London, UK), who reported that activation of either the urokinase-type plasminogen activator receptor (uPAR) or the epidermal growth factor receptor (EGFR) can induce the formation of abundant filopodia and membrane ruffles, as well as stimulating chemotaxis, in the metastatic mammary carcinoma cell line MDA-MB-231. However, only the EGFR pathway is inhibited by the PI3K inhibitor LY294002, indicating that distinct pathways can lead to phenotypically similar morphological outcomes. Bacterial and viral pathogens are particularly useful models in which to study signaling, as they hijack cellular processes to their own advantage and may circumvent normal regulatory circuits. Listeria monocytogenes is an invasive bacterium that enters cells using the host actin cytoskeleton, but uses it in different ways depending on cell type. P. Cossart (Paris, France) described how the bacterial surface protein internalin (InlA) binds E-cadherin on the surface of epithelial cells and, in a process requiring α-catenin, induces actin polymerization which allows bacterial internalization into the host cell. To gain entry to other cell types, another surface protein, InlB binds to the host gC1qR protein, also stimulating actin polymerization to gain entry, but in an alternative process that requires activation of PI3K. This phenomenon can be mimicked by adding InlB-coated beads to cells: ruffling is induced and the internalized beads become surrounded by actin and components of the Arp2/3 complex (see below). This simple system should be very useful in the dissection of actin assembly during phagocytosis. Downstream pathways: regulation of actin So what do these signal transduction pathways regulate? The last few years have seen many studies of the Arp2/3 complex, a seven polypeptide complex that is conserved from yeast to mammals, and that provides localized nucleation of actin assembly. L. Machesky (Birmingham, UK), who discovered the complex in Acanthamoeba some years ago, brought the meeting up to date on how it is regulated. In vitro, the Arp2/3 complex is a very poor nucleator of actin polymerization. The C-terminal region of proteins of the WASP (Wiskott-Aldrich-Syndrome protein) family (WASP/N-WASP/Scar1/2/3) contain a G-actin binding site (W domain) and an acidic (A) region that binds the p21 subunit of Arp2/3, and can activate the complex. However, full-length WASP proteins are poor positive regulators until they are locally activated by signals, such as those provided by the Rho GTPases or PIP2 (phosphatidylinositol 4,5-bisphosphate). The activated Arp2/3 complex induces the formation of dendritic F-actin structures by binding to the sides of existing filaments and nucleating new filaments with free barbed ends. By over-expressing a dominant-negative construct consisting of the WA region of Scar, Machesky showed that this mechanism seems to be responsible for actin assembly of stress fibers, for assembly at the protrusive leading edge of migrating cells, during phagocytosis, and in the motility of several intracellular pathogens. M.-F. Carlier (Gif-sur-Yvette, France) reported that the activated Arp2/3 complex branches filaments from the barbed ends in an autocatalytic fashion. Contrary to current views of Arp2/3 function, Carlier proposed that it does not act as a nucleator that stabilizes actin dimers, or as a filament pointed-end capper, or cause branching from the sides of actin filaments. In addition to the Arp2/3 pathway for actin polymerization, E. Friederich (Paris, France) provided evidence for a distinct mechanism of actin assembly. She described experiments in which a domain of zyxin was targeted to mitochondria, where it recruited VASP and was able to induce actin polymerization as shown by incorporation of rhodamine-labeled actin in premeabilized cells. Surprisingly, Arp3–GFP was not recruited in this system, suggesting the involvement of an alternative actin assembly pathway that does not use the Arp2/3 complex. Profilin has been suggested as an important regulator of actin turnover, and to be negatively regulated by PIP2. Results of structural studies described by S. Almo (New York, NY) included the finding that the profilin polypeptide may unfold upon binding PIP2 in vitro, raising the question of the physiological significance of the previously documented PIP2/profilin interaction, or perhaps uncovering a new form of protein regulation. Molecular motors The study of microtubule- and microfilament-based molecular motors has exploded into its own sub-field, and this area was represented by discussions of conventional kinesins and myosin Vs. F. Gyoeva (Moscow, Russia) discussed biochemical studies that showed that not all kinesin heavy chains have associated light chains, and that the heavy chain kinesin dimers always pair with light chains of the same kind. M. Schliwa (Munich, Germany) described the elegant use of Neurospora crassa, a filamentous fungus, in which alterations to the single non-essential kinesin generate distinct growth and morphological phenotypes. Using this system to assess functional domains, Schliwa presented results indicating that the C-terminal domain of kinesin interacts with and regulates the N-terminal motor domain. A highlight of the meeting was a dynamic presentation by T. Yanagida (Osaka, Japan) in which he first delighted the audience with movies of single kinesin molecules walking down a microtubule. Yanagida then discussed his recent studies on brain myosin V. These motors, perhaps the best studied of the unconventional myosin family, move down an actin filament in a processive manner to deliver various cargos. Myosin Vs have a long so-called lever arm, consisting of six IQ motifs. In a widely held view of the mechanochemical cycle of myosins, the step size is believed to be determined by the length of the lever arm–in particular, myosin V has been shown to have a large step size of ∼36 nm. Yanagida presented evidence, from experiments using optical trap nanometry, that a myosin V construct lacking five of its six IQ motifs moves with the same velocity and large step size as the wild type protein! Since both long and short lever arm myosin Vs have the same step sizes, Yanagida argued that the length of the arm cannot be the major determinant of step size and suggested that the head domain may make several continuous and short steps along the filament. He suggested that the large apparent step size of the myosin V might simply be determined by the helical cross-over length (36 nm) of the actin filament. The functions of an essential myosin V, encoded by the budding yeast MYO2 gene, were described by A. Bretscher (Ithaca, NY). Previous work had shown that the myosin moves along polarized actin cables, for example concentrating in a newly formed bud. By analyzing conditional mutations in the tail, Bretscher showed that this myosin V binds to and delivers secretory vesicles to sites of cell growth, as well as binding to the Kar9p/Bim1 complex on the ends of cytoplasmic microtubules early in the cell cycle to orient the nucleus in preparation for mitosis. Extending these observations to aspects of yeast cell polarization, Bretscher proposed that actin cables are major polarizing structures in yeast, and that the field is reaching a point where it is now possible to design and test models of how the entire microfilament system in yeast is established, how it functions and how it is regulated. Cell locomotion Cell locomotion is an integrated process, involving signal transduction pathways that coordinate protrusion by actin assembly at the front of the cell with adhesion formation at the front and disassembly at the rear, and with forward movement of the cell mass. F. Gertler (Cambridge, MA) described studies on the role of the Mena/Evl/VASP family of proteins in cell locomotion, making use of the fact that all members of the family contain an EVH1 domain that binds the sequence -DFPPPP-. By targeting copies of this sequence to mitochondria, all members of the family can be sequestered there and removed from their normal sites of action. Surprisingly, these cells show enhanced motility, as do cells derived from Mena/VASP knockout mice. In contrast, over-expression of Mena–GFP reduces movement and, at high levels of expression, also reduces cell polarity. Analysis of additional constructs and time-lapse photography of cells expressing them led Gertler to propose that the Mena protein family reduces the turnover of advancing and retreating areas of migrating cells. This is a remarkable result, as biochemical studies, especially those involving the mechanism of the intracellular movement of Listeria, had led to the prediction that the Mena/VASP proteins should accelerate actin-based cell motility. V. Small (Salzburg, Austria) showed a series of informative movies of migrating cells that illuminated the role of microtubules in polarizing the actin-based motile processes. It is well established that disassembly of microtubules in fibroblasts inhibits locomotion of fibroblasts by disrupting cell polarization. Small described how fibroblasts in which microtubules had been depolymerized could then be experimentally repolarized by using a micropipette to administer a local concentration of the myosin-light chain kinase inhibitor ML-7. The cells polarized away from the inhibitor, and when the inhibitor was continuously applied to the trailing edges, the fibroblasts were effectively ‘chased’ around the coverslip. This illustrated that even cells lacking microtubules have the intrinsic ability to polarize and migrate. So how do microtubules induce polarization and locomotion in untreated cells? Based on this and earlier work, Small presented a model whereby microtubules promote the turnover of cell-substratum adhesion sites by delivering signals that modulate actin–myosin contractility specifically at stress fibre termini. The key to the process seems to be restricting these modulatory effects of microtubules to specific regions, thereby regulating substrate adhesion patterns and, ultimately, cell polarity. Genetic approaches A powerful method to assess the particular roles of individual cytoskeletal components is genetic analysis, and the value of this approach was illuminated by presentations on studies performed in organisms ranging from yeast to mammals. D. Drubin (Berkeley, CA) described elegant studies in Saccharomyces cerevisiae that have elucidated the molecular interactions between various components of the actin-containing cortical patches that are distributed in a polarized fashion in yeast. Starting with a biochemically identified actin binding protein (Abp1p), he has used genetic approaches to uncover an amazing set of interactions between cortical patch components that range from actin to proteins of the clathrin coated vesicle endocytic pathway. Analysis of yeast mutants lacking these components has also indicated a function for the cortical patches in endocytosis. Moreover, mHip1R (related to Huntingtin-interaction protein), the mammalian homologue of the yeast Sla2p cortical patch protein, localizes to coated pits in cultured cells, underscoring the similarity of the yeast and mammalian systems. Another set of components of cortical patches that was identified by Drubin comprises the Ark1p/Prk1p kinases. Through the use of a kinase mutant that is sensitive to a specific inhibitor, Drubin demonstrated that these proteins regulate the structure and location of the cortical patches. S. Ono (Atlanta, GA) described genetic studies on cytoskeletal components in the nematode Caenorhabditis elegans. He found that mutations in muscle-specific cofilin (UNC-60B) or in an actin-interacting protein 1 (UNC-78) affect the dynamics of actin filaments during muscle development, giving rise to highly disordered filament aggregates. Finally, D. Louvard (Paris, France) and W. Witke (Monterotondo, Italy) described how genetic studies in the mouse are illuminating unexpected functions of actin binding proteins. Louvard reported on studies of mice lacking villin, a Ca2+-regulated actin-bundling/severing/capping protein which is normally a major component of intestinal epithelial brush border microvilli. Contrary to all expectations, loss of villin had no observable effect on the structure or function of brush border microvilli in mice. However, the mice were more susceptible to intestinal abrasive insults because they lacked epithelial cell plasticity and were therefore unable to repair cell injury. Moreover, inducible expression of villin in MDCK cells enhanced their motility in response to scatter factor. Witke summarized studies on mice lacking either gelsolin, another Ca2+ regulated actin severing/capping protein, or the related capping protein CapG. Remarkably, these knockout mice are viable in a mixed genetic background. However, when these mice are crossed to the Balb/c background, a genetic difference is uncovered revealing a requirement for gelsolin during embryogenesis. Other highlights Although the meeting was weighted towards microfilaments, other systems were represented as well. For example, M. Steinmetz (Basel, Switzerland) described elegant structural studies on the mechanism whereby stathmin/Op18 destablizes microtubules. Steinmetz presented a model in which stathmin binds two GTP-αβ tubulin dimers on the microtubule plus end and enhances their intrinsic GTPase activity, thereby acting as a ‘catastrophe’ factor. U. Aebi (Basel, Switzerland) presented his progress on studying the assembly of vimentin intermediate filaments by studying the X-ray structures of assembled subdomains. F. Ramaekers (Maastricht, The Netherlands) discussed studies on imaging GFP–lamins, which allowed him to visualize vertical tubules extending from one side of the nuclear lamina to the other. F. Braet (Brussels, Belgium) presented spectacular images of endothelial cells in which he showed that disruption of the actin cytoskeleton with various drugs greatly increased the number of fenestrae, and that treatment with the barbed-end actin filament capping drug misakinolide, for example, revealed a spiral fenestrae-forming center. C. Ampe (Ghent, Belgium) unveiled the biochemical mechanisms that allow actin and tubulin to be folded correctly by prefoldin and the chaperonin CCT. P. Meraldi (Martinsried, Germany) discussed the role of the Nek2 kinase and its potential substrate, C-Nap-1, in centrosomal separation and the role of E2F and Cdk2/cyclin A in controlling centrosome duplication. M. Bornens (Paris, France) also discussed the centrosome life cycle, visualized with GFP–centrin. He illustrated that, after telophase, the mother centriole remains near the cell center while the daughter centriole migrates around the cytoplasm in an actin- and microtubule-dependent manner. Conclusions Two decades ago, attempts to link cytoskeletal elements to essential cellular functions were often less than compelling. In the meantime, closer scrutiny has revealed that the cytoskeleton is the target of signal transduction pathways, playing organizing and integrating roles in multiple cell biological processes. On reflection, this is to be expected, as a cell has to be organized into a coherent whole, and the cytoskeleton seems to be the logical structure with which to do this. Perhaps G. Gabbiani and colleagues foresaw this development 20 years ago when they established the organization that grew into the European Cytoskeletal Forum. These important meetings will surely continue to enlighten us with new discoveries about the ways in which the cytoskeleton is integrated into cellular functions. The Forum will reassemble in Crete next year for their 16th Meeting, to hear and discuss the latest advances (see: http://www.weizmann.ac.il/eurocyto/home.html). Biography Anthony BretscherThe author is at the Department of Molecular Biology and Genetics, Biotechnology Building, Cornell University, Ithaca, NY 14853, USA References Bear J.E., Loureiro J.J., Libova I., Fässler R., Wehland J. and Gertler F.B. (2000) Negative regulation of fibroblast motility by Ena/VASP proteins. Cell, 101, 717–728.CrossrefCASPubMedWeb of Science®Google Scholar May R.C., Caron E., Hall A. and Machesky L.M. (2000) Involvement of the Arp2/3 complex in phagocytosis mediated by FcgammaR or CR3. Nature Cell Biol., 2, 246–248.CrossrefCASPubMedWeb of Science®Google Scholar Lecuit M., Hurme R., Pizarro-Cerda J., Ohayon H., Geiger B. and Cossart P. 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